Soil bacteria belonging to the genus Azotobacter can reduce atmospheric nitrogen into ammonia, which can be assimilated by plants.
About 20 genes are involved in the process of nitrogen reduction. Expression of all these genes is dependent on a positive regulator
NifA. In the presence of ammonia, the negative regulator NifL, gets activated and interacts with NifA to neutralize it. As a result no
nitrogen is reduced by wild type Azotobacter when ammonia is formed or produced by nitrogenous fertilizers. In two species of
Azotobacter studied by us, the nifL and nifA genes were present in the same operon, nifL being proximal and nifA distal to the promoter.
The nifLA promoter was also regulated by ammonia. We partially deleted the nifL gene and inserted a constitutive promoter there, thus
bringing the nifA gene under its control. Ammonium only marginally affected acetylene reduction (a measure of nitrogen reduction)
by the engineered Azotobacter strain. When wheat seeds were inoculated with the engineered strain and sown, the crop yield was
enhanced by 60%, without any application of urea or other chemical nitrogenous fertilizer. The crop yield, however, was enhanced only
by 10% when the wheat seeds were inoculated with the wild type strain. When we applied urea, the wheat plants arising from the seeds
inoculated by the engineered strain, could yield the same amount of crop using ~85 kg less urea (~40 kg less nitrogen) than the usual
~257 kg urea (~120 kg nitrogen) per hectare. Wheat plants from the seeds inoculated with the engineered strain had much higher dry
weight and nitrogen content and assimilated molecular 15 N much better than plants from seeds inoculated with the wild type strain.
Inoculation of the wheat seeds with the engineered strain did not adversely affect the microbial population in the rhizosphere soil.